For more than a decade, the development of synthetic probes for the recognition and analysis of sugars has attracted much attention. Synthetic probes could find useful applications in the food industry as well as in the clinical analysis. Detection and monitoring of glucose is particularly important for diabetics. The use of enzymes shows some limitations in the development of implantable sensors for continuous glucose monitoring in blood or interstitial tissue. Continuous monitoring of glucose blood level is very important for the long term health of the diabetics and could lead to important medical technology such asa blood sugar alarm system and an in vivo control device for an implanted insulin pump.
The boronic acids have been known for their ability to interact with diols (1). In addition, boronic, arsenious, germanic and telluric acid derivatives are known to exhibit similar characteristics (See e.g., U.S. Pat. No. 5,512,246). These compounds have been used for the development of receptor and fluorescent probes for sugars (2-4). One advantage of using boronic acid as a chelator group for sugars is the compound's fast and reversible interaction with sugars. In addition, many substituted phenylboronic acids are commercially available, which would allow for the development of a large diversity of synthetic fluorescent probes for sugars with minimal synthetic steps. Depending on the structure of the molecule and on the number of boronic acid group present, association constants from micromolar to tens of millimolar can be obtained and chiral discrimination can also be observed. Delivery systems for insulin have also been developed using boronic acid gel (5-6).
Determination of the glucose concentration is crucial for people with diabetes. Large variation in the glucose level in blood could result in important medical problems including cardiovascular disease, neuropathies and blindness. Non-invasive measurement of blood glucose has been a long-standing research goal and a wide variety of such methods have been describe in the literature, including near-infrared spectroscopy, optical rotation, amperometric, calorimetric, and fluorescence detection (7-20). Despite some promising results, these methods show limitation as important background with the NIR (Near infra red) technique and low optical rotation and important depolarization du to the tissue with the optical rotation technique. Enzymes and proteins are widely use in the research for the development of glucose sensors. At present, the most reliable method to measure blood glucose is by finger stick and subsequent glucose measurement, typically by glucose oxidase. A competitive glucose assay using fluorescence resonance energy transfer between concanavalin A and dextran has been developed and efforts are also underway to develop methods for the use of intrinsic fluorescence changes using thermophilic enzymes. Proteins and enzymes show an affinity constant comparable with blood glucose level, show a great selectivity and are biocompatible. Despite these advantages, they exhibit low stability (to heat and organic solvents), solubility problems and are difficult to modify. Thus, the development of a synthetic glucose sensor is greatly desirable and the flexibility of organic compounds as probes could allow a wide range of possibilities for the development of a non-consuming glucose device.
In treating diabetic patients, the aim is to tightly regulate the plasma glucose level within the normal physiological range (80-120 mg/dL), so that diabetic adverse effects can be avoided. As an aid to diabetes therapy, continuous monitoring of blood glucose concentrations in vivo has long been recognized as a major objective as a future tool in the fight against diabetes. During the past decade, intense effort has been directed toward the development of glucose monitoring biosensors as an aid to diabetes therapy. Development of an implantable glucose sensor that is specific to glucose and sensitive enough to precisely measure glucose levels in vivo would be a significant advance in the treatment of diabetes. Such ability to more closely control blood glucose levels would also be useful in insulin delivery system responsive to glucose levels in diabetic patients. Glucose biosensor systems have recently been described which employ glucose binding molecules attached to a polymeric hydrogel for example (See. e.g., U.S. Pat. No. 6,475,670).
For several decades, fluorescence spectroscopy has been widely use for the detection and analysis of different analytes (20-22). Wavelength-ratiometric, fluorescence lifetime based sensing and polarization assays (24-26) are some techniques available for the detection and analysis of analytes by fluorescence spectroscopy. Fluorescence techniques for glucose recognition have been used most of the time with enzymes and proteins. Despite some promising results, enzymes and proteins show some stability problems against organic solvents and heat. In contrast, synthetic organic probes show high stability and flexibility due to the versatility of the organic synthesis. Modification of the probe structure could lead to a modification of the affinity for the analyte, of the wavelength of emission of the probes and of the immobilization of the probes on a support for the building of a sensor.
The use of the intramolecular charge transfer (ICT) involving the boronic acid is a very promising technique for rapid monitoring of sugar levels. ICT is well known to be very sensitive to small perturbations that can result in spectral shifts, intensity changes and/or lifetime changes. In addition, ICT can be applied to a large diversity of fluorophores without limitation of the wavelength range and/or lifetime of the fluorophore. The boronic acid group has been known for 40 years for the ability to form complexes with polyols. This ability led Yoon et al. to build a fluorescence probe for sugar based on the boronic acid group. S. Shinkai, T. D. James and collaborators have developed and studied some molecular structures and fluorescence probes involving the boronic acid group (27-33). They have developed fluorescence probes involving different mechanisms to induce spectral changes. Molecular rigidification, photoinduced electron transfer (PET) and excimer formation are some examples (34-36). Despite these interesting studies, most of the fluorescence probes developed up to now show emission in the ultraviolet region and/or involved a mechanism limited to few fluorophores.
Photoinduced electron transfer (PET) is often used as mechanism for fluorescence quenching in the development of sensors. This quenching is due to the presence of the amino group near the chromophore. When an analyte (ions for almost all cases) binds the probe, the interaction between the analyte and the nitrogen's lone pair of electrons removes the quenching and results in a detectable increase of the fluorescence of the probe. This mechanism has been applied with an anthracene derivative with amino and phenyl boronic acid groups for glucose probes. Upon the binding between the boronic acid group and the saccharide, the pKa of the boron atom decreases. This decrease improves the interaction between the boron atom and the nitrogen atom of the amino group and thus reduces the PET quenching of the chromophore. Increase of the fluorescence intensity up to seven time can be observed. These anthracene probes for saccharide have also been successful adapted to build polymers for the development of a device (37-38). Until now, however, no system and analysis using the fluorescence lifetime could be found for these systems.
Recent interest in the boron-aromatic systems stems from the concept of π-electron aromaticity and conjugation across sp2-hybridized boron. Recent reports highlight the potential use of boron-containing conjugated polymers in the emerging optoelectronic applications. Lee et al. investigated the effect of the B−-for-C substitution on the photophysics and photochemistry of borastilbenes and borastyrylstilbenes (39-40). The phenyl boronic acid group [phe-B(OH)2] has attracted interest for its ability to covalently bind diols and sugars. Lorand et al. investigated the structure of the neutral and anionic forms [phe-B(OH)3−] of the phenylboronic acid group. Their results showed that the neutral form of the boronic acid group linked to the phenyl moiety has a planar triangular conformation with a sp2-hybridized boron atom. On the other hand, the anionic form has a tetrahedral conformation with a sp3-hybridized boron atom. Two research groups have investigated the effect of this change on the emission of fluorophores in order to evaluate their use for the development of fluorescent probes for saccharides. Yoon et al. examined anthrylboronic acid and Suenaga et al. analyzed naphthyl, biphenyl, pyrenyl and stilbeneboronic acid. In the case of the anthrylboronic acid, a decrease of 40% of the emission intensity was observed following the formation of the anionic form of the boronic acid group. Complexation of the boronic acid moiety with saccharides decreases the pKa of boronic acid group, 8.8 to 5.9 in saturated fructose solution. As a result, complexation with the saccharide induces the formation of the anionic form of the boronic acid and then a decrease of the emission intensity. This decrease is relatively small, 30% for fructose and about 10% for glucose. Suenaga et al. observed similar results. For this reason, the direct insertion of the boronic acid group on a fluorophore has not been deeply investigated.
Several laboratories have investigated the ability of the boronic acid group to interact with amino groups. Fluorescence probes based on a decrease of the photoinduced electron transfer (PET) of amino-substituted fluorophores, mainly anthracene, have been synthesized (42). This mechanism resulted in a significant intensity increase, up to 7-fold, and a fluorescence lifetime change after binding saccharides. Molecular rigidification induced by saccharides interaction using the boronic acid group as a chelator group has also been used with a cyanine dye for the development of fluorescence probes. Excimer formation between two pyrene moieties has also been used. Despite these interesting approaches to use the combination of the boronic acid group and fluorophores, they are mostly restricted to a few fluorophores. The PET mechanism is expected to be ineffective for long wavelength fluorophore use (43). Rigidification and excimer formation can be applied only to few fluorophores.
One objective of the invention is to provide compounds which are useful as fluorescent probes for the detection of sugars. Another objective of the invention is to provide fluorescent probes that are useful for the detection of sugars based on lifetime fluorescence, changes in fluorescent intensity, spectral shifts and/or wavelength-ratiometric measurements.
The present invention fulfils these needs and realizes these and other objectives. Other advantages of the invention are further apparent from the disclosure provided.